Antibacterial Activities of Polyethylene Glycol, Tween 80 and Sodium Dodecyl Sulphate Coated Silver...

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Copyright © 2012 American Scientific PublishersAll rights reservedPrinted in the United States of America

Journal ofNanoscience and Nanotechnology

Vol. 12, 2513–2521, 2012

Antibacterial Activities of Polyethylene Glycol,Tween 80 and Sodium Dodecyl Sulphate CoatedSilver Nanoparticles in Normal and Multi-Drug

Resistant Bacteria

Debalina Bhattacharya1, Saheli Samanta1, Ananda Mukherjee1, Chitta Ranjan Santra2,Amar N. Ghosh3, Swapan Kumar Niyogi3, and Parimal Karmakar1�∗

1Department of Life Science and Biotechnology, Jadavpur University, 188, Raja S.C. Mullick Road, Kolkata 700032, West Bengal, India2Department of Chemistry, Netaji Nagar Day College, NSC Bose Road, Regent Estate, Kolkata 700092, West Bengal, India

3National Institute of Cholera and Enteric Diseases, P-33 CIT Road, Scheme XM, Kolkata 700010, West Bengal, India

Antibacterial activity of silver nanoparticles coated with different functionalizing agents i.e., polyethy-lene glycol, tween 80 and sodium dodecyl sulphate were evaluated on both normal and multi-drugresistant strains of bacteria. Under the same reaction conditions, these functionalizing agents wereadded separately to coat silver nanoparticles. Among these, polyethylene glycol coated nanopar-ticles were most effective in killing all the bacterial strains which includes Escherichia coli DH5�,Bacillus subtilis, Micrococcus luteus, Staphylococcus aureus and multi-drug resistant clinical iso-lates of Shigella spp. (flexneri, boydii, sohnea) and Vibrio cholerae. The minimum inhibitory con-centration of polyethylene glycol coated silver nanoparticles was also less compared to the othertwo sets of nanoparticles. Consistence with that polyethylene glycol coated nanoparticles producedmore intracellular reactive oxygen species in bacteria. Moreover, when human cell lines MCF7 andChang Liver were incubated in presence of these nanoparticles for 18 h with same concentrationsas used for bacteria, no toxicity was observed. But significant increase in cell killing was observedwith longer incubation time. Thus our present investigation implicates the potential therapeutic useof silver nanoparticles as antibacterial agent particularly the polyethylene glycol coated one.

Keywords: Silver Nanoparticles, Multi-Drug Resistant Bacteria, Antibacterial Activity, MinimumInhibitory Concentration, Reactive Oxygen Species.

1. INTRODUCTION

There is a continuous search for new generation of antibi-otics to counteract the bacterial infection due to frequentoccurrence of antibiotic resistant bacteria. In past fewdecades several agents have been synthesized to counteractthe antibiotic resistant bacterial strains. Among these, sil-ver nanoparticles (AgNPs) are one of the promising agentsfor several reasons.1�2 They are effective on both Grampositive and Gram negative bacteria. They are also effec-tive against virus3�4 and fungus.5�6 Moreover, silver has alower propensity to induce microbial resistance than manyother antimicrobial substances.7–10 The use of silver asantimicrobial agent has been known for century. Recentdevelopment of nanotechnology opens up the opportunity

∗Author to whom correspondence should be addressed.

to revisit their antimicrobial activity and research beingcarried out to develop suitable therapeutic agents withAgNPs, in particular to combat antibiotic resistant bacteria.In such efforts, several techniques have been developed tomodify AgNPs with different agents to achieve better bio-logical activity such as carbon nanotubes,11�12 amphiphilicmacromolecules,13 chitosan,14�15 silica nanospheres,16�17

and TiO218�19 etc.

Chemical synthesis of AgNPs by reducing silver saltswith sodium borohydride (NaBH4� is one of the simplestmethods. To increase stability, solubility and to pre-vent aggregation, functionalizing agents like water solu-ble polymers, oligosaccharides and polysaccharides, SDSand sophorolipid19�20 etc. are generally used for effi-cient binding with AgNPs. The extent of antibacterialeffect of AgNPs is supposed to depend on size, shape,agglomeration21 and nature of the functionalizing agents.22

J. Nanosci. Nanotechnol. 2012, Vol. 12, No. 3 1533-4880/2012/12/2513/009 doi:10.1166/jnn.2012.6148 2513


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Antibacterial Activities of PEG, T80 and SDS Coated Silver Nanoparticles in Normal Bhattacharya et al.

The mechanisms of action of AgNPs on bacterialcells remain unknown. It has been proposed that beingextremely large surface area AgNPs may have betterchance to contact with bacteria. Ag+ ions released byAgNPs are proposed to be responsible for their enhancedantibacterial activity.23�24 It has been suggested that AgNPstarget bacterial membrane proteins and interfere with pro-ton motive force.25 Furthermore, AgNPs may attack res-piratory chain of bacteria and block cell division leadingto bacterial cell death.1 Using proteometric analysis it hasbeen reported that AgNPs can up regulate or down regu-late several key enzymes for the endurance of bacteria.26

On the other hand Ag+ ions have better chance to inac-tivate various enzymes inside the cells.27 Thus, AgNPseffects are likely to be distinct from those of Ag+ ionsalone and rather depend on size and nature of coating.It has been also proposed that effects of AgNPs are notsimply due to the release of Ag+ ions into the surround-ing environment.26�27 Thus taken together it seems thatmechanism of action of AgNPs remain unknown and maydepends on various factors including the agents used fortheir stability.Though there is one report, which documents that sil-

ver exhibits low toxicity to mammalian cells,28 there areseveral reports about the cytotoxic effects of AgNPs ondifferent cells. AgNPs can induce DNA damage and apop-tosis in a dose dependent manner.29�30 Deregulation of dif-ferent genes including DNA repair genes by AgNPs havealso been documented by micro-array assay.30 Apart fromthe presence of Ag+ ions,28 AgNPs are also reported toinduce oxidative stress.31�32 Thus, optimization of AgNPsby regulating their synthesis, stability and concentrations,are necessary for using them as antimicrobial agents whichwill not perturb normal activity of human cells.With this view, we used three functionalizing agents

polyethylene glycol (PEG), tween 80 (T80) and sodiumdodecyl sulphate (SDS) to stabilize AgNPs during theirchemical synthesis. Surface plasmon resonance (SPR)and Fourier transform infrared spectroscopy (FTIR) wereused to confirm their functionalization. Further their sizesand shapes were determined by Dynamic light scatter-ing (DLS) and Transmission electron microscopy (TEM).Wide range of bacteria including multi-drug resistant(MDR) strains have been tested and we observed that PEGcoated AgNPs were most effective in killing all the bac-terial strains. Further Minimum inhibitory concentration(MIC) and Minimum bactericidal concentration (MBC) ofPEG coated AgNPs were minimum among the three. Fromgrowth curve analysis it was confirmed that all AgNPswere bactericidal rather than bacteriostatic. Moreover, withthe concentrations range as used in bacteria, we demon-strated that all the three AgNPs were non toxic to twohuman cell lines, human breast cancer cell line, MCF7and normal human liver cell line, Chang Liver. So, amongthe three AgNPs we have tested, PEG coated AgNPs have

maximum potential to kill bacteria and may have thera-peutic implication in future.

2. MATERIALS AND METHODS

2.1. Reagents

AgNO3, NaBH4 and PEG of the highest purity were pur-chased from Scientific Research Laboratory (SRL). T80and SDS were purchased from Sigma. LB agar and nutri-ent agar medium were purchased from Himedia.

2.2. Bacterial Strains and Growth Conditions

Gram negative bacteria E. coli DH5� (MTCC 1652 India)and three Gram positive bacteria S. aureus (MTCC 96),M. luteus (MTCC 106) and B. subtilis (MTCC 441)and two artificially transformed bacterial strains E. coliDH5�/Puc19 (AmpR� and E. coli DH5�/DS-Red (KanaR)were grown, sub cultured and maintained on LB mediumand stored at 4 �C. Two clinically isolated MDR strains ofV. cholerae O1 (C9535 and B8779) and two MDR strainsof Shigella flexneri (IDH00177 and IDH00178), two MDRstrains of Shigella boydii (IDH00635 and IDH00646)and two MDR strains of Shigella sonnei (IDH00734 andIDH00741) were grown on nutrient agar medium andstored at room temperature.

2.3. Preparation of PEG, T80 andSDS Coated AgNPs

AgNPs were prepared by the chemical reduction ofAgNO3 by NaBH4. The normal reduction process was per-formed according to the standard protocol.33 The coatingsof AgNPs were done in presence of various functional-izing agents (PEG, T80 and SDS) individually keepingAgNO3 and NaBH4 concentration same. Then these coatedAgNPs were purified by centrifugation. To remove excessAg+ ions and excess functionalizing agents, the pellet waswashed three times with deionized water. A dried pow-der of coated AgNPs was obtained by freeze drying. Forfurther experiments the powder was resuspended in deion-ized water, and the suspension was homogenized with anultrasonicator (Sartorius).

2.4. Physical Characterization of PEG,T80 and SDS Coated AgNPs

The synthesized nanoparticles were characterized by var-ious methods like DLS, TEM, UV/Visible spectroscopyand FTIR. The average particle size i.e., hydrodynamicdiameter (dh� was evaluated by DLS using Nano-ZS(Malvern) instrument (5 mW HeNe laser = 632 nm) at25 �C. To determine the morphology of coated AgNPs,samples were prepared after drying on carbon coated cop-per grid and observed under a TEM (FEI, Model: TECNAI

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Bhattacharya et al. Antibacterial Activities of PEG, T80 and SDS Coated Silver Nanoparticles in Normal

12 Bio Twin) with an accelerating potential of 100 kVand mean particle size was analyzed with ANALYSISsoftware. UV/Vis absorption spectra of PEG, T80, andSDS coated AgNPs were acquired in 300–800 nm wave-length range using an UV/Vis spectrophotometer (HitachiU-2800) operating at a resolution of 2 nm. FTIR spec-tra were measured in 4000–400 cm−1 range with a res-olution of 1 cm−1 using a Perkin-Elmer Spectrum GXinstrument in transmission mode using potassium bromide(KBr) pellets.

2.5. Antibacterial Activity Measurement

This assay was done by standard agar well diffusionmethod. All the strains were grown on LB broth at 37 �Cup to a turbidity of 0.5 Mac Farland standards (1.5×108 CFU/ml). About 100 �l of this suspension was usedto inoculate 90 mm diameter petridish filled with 35 ml ofLB agar. Wells (diameter2 = 0.563 cm2� were punched inthe agar plates and filled with 100 �l of coated AgNPs.Plates were incubated at 37 �C for overnight. Antibacterialactivities were evaluated by measuring the area of zoneof inhibition (diameter2). Sterile water and functionalizingagents were used as negative control.34

2.6. Determination of MIC andMBC of Coated AgNPs

MIC and MBC were determined by micro dilution method,using an inoculum of 106 CFU/ml of both Gram posi-tive and Gram negative bacteria (normal and MDR strains)in separate tube along with increasing concentrations ofAgNPs. The cultures were incubated at 37± 2 �C andshaken at 200 rpm. Bacterial cell viability was determinedby measuring OD at 600 nm. Briefly, after incubation,MBCs were determined by plating 100 �l of each sampleon the LB agar plate. MIC value indicates the concentra-tion which inhibits 99% of bacterial growth, while MBCvalue indicates the concentration which inhibits 100% ofbacterial growth, both compared to the positive control(mock treated).35�36

2.7. Bacterial Growth Kinetics Measurement

To examine the bacterial growth kinetics in presence ofPEG, T80 and SDS coated AgNPs, E. coli DH5� andM. luteus cells were grown in 100 ml LB broth in presenceof all the three AgNPs separately, at a dose lower thanthe corresponding MIC value at 37 �C with continuousshaking at 200 rpm. Bacterial concentrations in presenceof coated AgNPs were determined by measuring OD at600 nm in every 60 mins interval. Antibacterial kineticswas plotted graphically with time versus OD600.

22

2.8. ROS Assay by Nitroblue Tetrazolium(NBT) Reduction

0.1 ml bacterial suspension (where OD600 = 1�0) in Hanksbalanced salt solution (HBSS) was incubated with respec-tive AgNPs and 0.5 ml of 1 mg/ml NBT for 30 mins at37 �C. Then 0.1 ml of 0.1 M HCl was added and tubeswere centrifuged at 1500 g for 10 mins. The pellets weretreated with 0.6 ml DMSO to extract the reduced NBT;finally, 0.5 ml HBSS was added and OD was measured at575 nm (intracellular ROS).37

2.9. Cell Culture

Human normal liver cells (Chang Liver) and breast cancercells (MCF7) were cultured in DMEM and RPMI-1640media supplemented with 10% fetal bovine serum and100 U/ml penicillin-streptomycin at 5% CO2 and 37 �C.

2.10. MTT Assay

Cells were seeded at a density 1× 104 cells/well in a96 well plates and after 24 h cells were exposed to respec-tive AgNPs at various concentrations as mentioned in thefigure legend for 18 h, 48 h and 72 h. After incuba-tion, cells were washed with PBS twice. Then cells werereplaced with MTT solution and incubated for 3–4 h at37 �C until a purple coloured formazan product developed.The resulting formazan product was dissolved in DMSOand OD were measured at 570 nm by using a microplatereader (Biorad) and the percentage survival was calculatedusing untreated cells as 100%.31

2.11. Statistical Analysis

All data were represented as mean±SD of three separateexperiments. Student’s t-test was used to determine statis-tical significance for multiple comparisons. P < 0�05 wasaccepted as statistically significant.

3. RESULTS

3.1. Physical Characterization of PEG,T80 and SDS Coated AgNPs

AgNPs were chemically synthesized by reducing silversalts with NaBH4 in presence of three different function-alizing agents PEG, T80 and SDS separately. After syn-thesis, each of the samples were extensively washed withdeionized water to remove excess Ag+ ions, NaBH4 andfunctionalizing agents. Then they were characterized bydifferent physical methods. DLS study showed the poly-dispersity of PEG coated AgNPs was 0.254 but in caseof T80 and SDS coated AgNPs the corresponding valueswere 0.227 and 0.285 respectively (data not shown).

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Fig. 1. SPR of coated AgNPs compared with bare AgNPs. There is aslight red shift from 410 nm to 420 nm for bare AgNPs to PEG, T80and SDS coated AgNPs.

(a)

(b)

(c)

Fig. 2. TEM of coated AgNPs. (a) AgNP PEG, (b) AgNP T80,(c) AgNP SDS.

Moreover, SPR study showed (Fig. 1) slight red shiftsafter the coating. Bare AgNPs showed a peak at 410 nm,but for coated AgNPs peak shifted to 420 nm indicat-ing the association of the said agents with AgNPs. Thatwas also confirmed by FTIR. The characteristic absorptionpeaks in FTIR were as follows: absorption peaks of SDS:C–O: 1081 (s); 1220 (s); 1249 (s) and C–H: 1379 (s);AgNP SDS: C–O: 1213 (s); 1267 (s) and C–H: 1380 (s),T80: C O: 1643 (m), 1735 (s); C–O: 1107 (s), 1249 (m),

(a)

(b)

Fig. 3. (a) Comparative study of the zone of inhibition (cm2) for coatedAgNPs in four normal bacterial strains. The data is the average ofthree experiments ± SD. ∗ = represents P value < 0�05, ∗∗ = representsP value < 0�01, ∗∗∗ = represents P value < 0�001, ns = representsP value > 0�05. (b) Size variation study of the zone of inhibition (cm)for coated AgNPs in E. coli DH5�. The data is the average of threeexperiments ± SD.



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Table I. Zone of inhibition (cm2) for PEG, T80 and SDS coated AgNPsin two artificially transformed bacterial strains (Ampr and Kanar).

Inhibitory Zone in Sq. Diameter (cm2�Name of thebacterial strain AgNP SDS AgNP Tween 80 AgNP PEG

E. coli DH5�/PUC19 2.46 ± 0.30 3.12 ± 0.16 3.74 ± 0.18(Ampr�

E. coli DH5�/DS-Red 2.15 ± 0.14 3.12 ± 0.16 3.74 ± 0.18(Kanar�

The data is the average of three experiments ± SD.

1298 (m); AgNP T80: C O: 1635 (m), 1733 (s); C–O:1107 (s), 1249 (m), 1298 (m); PEG: O–H: 3445 (s-Sharp),C–O: 1110 (s), 1147 (s), 1240 (m), 1280 (s); C–H bending1344 (s) 1359 (m); AgNP PEG: O–H: 3442.7 (s-broad),C–O: 1245 (m),1282 (s); C–H bending 1348 (w) 1379 (w).Bare AgNPs did not show any characteristic absorptionpeaks as usual. The FTIR spectra of pure PEG, T80,SDS and that of AgNP PEG, AgNP T80 and AgNPSDS showed no significant shift in stretching frequen-cies, which indicated that no direct bond formation tookplace between the functionalizing agents and the nanopar-ticles. But even after repeated washing of AgNPs withdeionized water the spectral pattern of pure functionalizingagents were retained, which clearly indicated that function-alizing agents were wrapping around AgNPs to stabilizethem. Perhaps Van der Walls force was acting between thenanoparticles and the stabilizing agents. The FTIR dataalso confirmed that during the reaction with NaBH4 noreduction of the functionalizing agents occurred.The exact size of the nanoparticles was determined

by TEM (Fig. 2). The average diameters of PEG coatedAgNPs were 14 nm, T80 coated AgNPs were 45 nmand for SDS coated the corresponding value was 54 nm.

Table II. Zone of inhibition (cm2) for PEG, T80 and SDS coatedAgNPs in MDR strains.

Inhibitory Zone in Sq. Diameter (cm2)Name of thebacterial strain AgNP SDS AgNP Tween 80 AgNP PEG

Shigella flexneri 1.78±0.13 2.35±0.15 3.12±0.162aIDH00178

Shigella flexneri 2.15±0.14 2.67±0.19 3.36±0.173aIDH00177

Shigella boydii 2.35±0.15 3.00±0.16 3.87±0.18IDH00635

Shigella boydii 1.78±0.13 3.00±0.16 3.36±0.17IDH00646

Shigella sonnei 2.06±0.26 2.67±0.19 3.74±0.18IDH00734


Vibrio cholerae 2.36±0.28 2.67±0.19 3.48±0.17C9535

Vibrio cholerae 2.35±0.15 3.12±0.16 3.36±0.17B8779


Table III. MIC values for E. coli DH5�, M. luteus, S. aureus andB. subtilis.

MIC (�g/ml) for 106 bacteria/mlName of thebacterial strain AgNP SDS AgNP Tween 80 AgNP PEG

E. coli DH5� 100±0.01 65.3±2.00 30±1.9(Gram –Ve)

S. aureus 116.5±1.00 83.25±0.001 23.31±0.03(Gram+Ve)

B. subtilis 131± .01 83.25±1.5 37.74±1.8(Gram+Ve)

M. luteus 100±1.5 66.6±0.05 30±0.01(Gram+Ve)

E. coli DH5�/Puc19 116.5±2.0 83.25±1.8 33.3±2.00(Ampr�

E. coli DH5�/DS-Red 133.2±1.5 98.6±0.03 33.3±1.7(Kanar�


Therefore, PEG coated AgNPs were smallest in size com-pared to the other two and there was no agglomeration. Allthe coated particles were stable for very long time (morethan six months) at room temperature.

3.2. Antibacterial Activity Measurements

Antibacterial activity of functionalized AgNPs was doneby agar well diffusion assay on four bacterial strainsi.e., E. coli DH5�, B. subtilis, S. aureus and M. lutues(Fig. 3(a)). Surprisingly M. lutues was most sensitive andB. subtilis was least sensitive among all the strains. ButPEG coated AgNPs were most effective for all the bac-terial strains tested. To explore whether AgNPs couldkill antibiotic resistant strains of bacteria, E. coli DH5�was transformed with plasmid containing ampicillin orkanamycin resistance gene separately and then subjected toagar well diffusion assay in presence of coated AgNPs. As

Table IV. MIC values for MDR strains of Shigella spp. and V. cholerae.

MIC (�g/ml) for 106 bacteria/mlName of theMDR Strains AgNP SDS AgNP Tween 80 AgNP PEG

Shigella flexneri 149.5±1.00 100±2.00 30±2.5IDH00178

Shigella flexneri 149.5±0.05 83.25±1.8 23.31±1.7IDH00177

Shigella boydii 100±0.01 96.6±3.8 39.96±0.07IDH00635

Shigella boydii 98.6±0.05 100±2.2 33.3±0.05IDH00646


Shigella sonnei 100±3.4 83.25±1.00 33.3±1.8IDH00741

Vibrio cholerae 129.4±0.01 100±0.05 37.74±1.00C9535

Vibrio cholerae 131±0.03 100±0.01 39.96±1.00B8779




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Table V. MBC of four normal bacterial strains.

Name of the MBC for AgNP MBC for AgNP MBC for AgNP MBC/MIC Ratio MBC/MIC Ratio MBC/MIC RatioStrains SDS (�g/ml) T80 (�g/ml) PEG (�g/ml) for AgNP SDS for AgNP T80 for AgNP PEG

E.coli DH� (Gram−Ve) 100±0.01 67.4±0.0 30±0.19 1.00 1.00 1.00S aureus (Gram+Ve) 118±0.001 83.25±0.001 23.31± .03 1.01 1.00 1.00B. subtilis (Gram+Ve) 133.3±0.01 85±1.5 37.74±1.8 1.01 1.02 1.00M. luteus (Gram+Ve) 110±2.5 66.6±0.05 30±0.01 1.1 1.00 1.00


shown in Table I, all the coated AgNPs were able to inhibitthe growth of these transformed bacteria. InterestinglyPEG coated AgNPs were also most effective in killing theantibiotic resistant strains. One step further, when we ana-lyzed some clinically isolated MDR strains, it was noticedthat all the coated AgNPs were able to inhibit their growth(Table II). Here also PEG coated AgNPs were most effec-tive in killing these strains. Next, we synthesized all thethree coated nanoparticles separately in different diametersranges from 20 nm to 90 nm and then determined theirantibacterial activity. As shown in Figure 3(b), antibacte-rial activity of PEG coated one was most effective com-pared to the other two except at 90 nm where antibacterialactivity of PEG coated and T80 coated AgNPs were equal.Surprisingly SDS coated nanoparticle at a diameter below50 nm had no antibacterial activity.

3.3. Determination of MIC and MBC

We next determined MIC of each set of nanoparticles forall the bacterial strains. As shown in Tables III and IV,PEG coated AgNPs required least amount compared tothe other two sets of nanoparticles. We further determinedMBC for each sets of nanoparticles. As shown in Table Vthe ratio between MBC and MIC did not change much(∼1, for all the cases). This indicated that all the coatedAgNPs were bactericidal rather than bacteriostatic agent.

3.4. Bacterial Growth Kinetics Measurement

Further analysis was carried out on the growth curve ofM. luteus and E. coli DH5� to check whether these AgNPscould kill or inhibit the growth of these bacteria. Boththe bacterial strains were exposed to all the three sets ofAgNPs separately, at concentrations lower than their cor-responding MIC values. As shown in Figures 4(a) and (b),the lag phase of all AgNPs treated bacteria were extendedand the growth of both the strains were mostly affected byPEG coated one.

3.5. ROS Assay

To understand the mechanisms of antibacterial activity ofAgNPs, intracellular reactive oxygen species (ROS) wasestimated. As shown in Figure 5, after addition of AgNPs,the production of ROS increased steadily with time. Thegeneration of ROS in case of PEG coated AgNPs was

increased 53.5% in 1 h compared to mock treated bacte-ria. Also ROS productions for other two sets of AgNPstreated bacteria were increased (42.7% in case of T80coated AgNPs and 37.3% in case of SDS coated AgNPs).Thus, PEG coated AgNPs produced maximum amount ofROS compared to the other two sets of AgNPs.

3.6. MTT Assay

For therapeutic applications, the effects of coated AgNPson human cells should be considered. To address that, wetested the effect of these nanoparticles on two human cell

(a)

(b)

Fig. 4. Growth curve of M. luteus (a) and E. coli DH5� (b) in presenceof PEG, T80 and SDS coated AgNPs.



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Fig. 5. Comparative study of ROS production of E. coli DH5� treatedwith of PEG, T80 and SDS coated AgNPs. The data is the aver-age of three experiments ± SD. The data is normalized from con-trol (without treatment) ∗ = represents P value < 0�05, ∗∗ = representsP value < 0�01, ∗∗∗ = represents P value < 0�001, ns = representsP value> 0�05.

(a)

(b)

Fig. 6. MTT Assay of MCF 7 (a) and Chang liver (b) with PEG, T80and SDS coated AgNPs. The concentrations are of 0 �g/ml, 3.4 �g/ml,6.8 �g/ml, 13.6 �g/ml, 20.4 �g/ml, 27.2 �g/ml, 34 �g/ml, 40.8 �g/ml,46 �g/ml respectively. Incubation time 18 h. The data is the average ofthree experiments ± SD.

(a)

(b)

Fig. 7. MTT Assay of MCF 7 for 48 h (a) and 72 h (b) with PEG, T80and SDS coated AgNPs. The concentrations are of 0 �g/ml, 3.4 �g/ml,6.8 �g/ml, 13.6 �g/ml, 20.4 �g/ml, 27.2 �g/ml, 34 �g/ml, 40.8 �g/ml,46 �g/ml respectively. Incubation time 48 h and 72 h. The data is theaverage of three experiments ± SD.

lines, MCF7 and Chang Liver. For these experiments, weapplied the same concentrations range as used in bacteria.As shown in Figures 6(a) and (b), no significant toxiceffects were observed in both the cell lines when theseAgNPs were treated for 18 h. But when cells were incu-bated in presence of the said agents for 48 h and 72 h,cytotoxicity was increased significantly. From Figure 7 theLD50 values for PEG coated and T80 coated AgNPs werecalculated to be 15 �g/ml and 22 �g/ml for 48 h and3.5 �g/ml and 6.8 �g/ml for 72 h respectively. Similarlyfor SDS coated AgNPs the LD50 value for 48 h was notsignificant and 10 �g/ml for 72 h.

4. DISCUSSIONS

AgNPs are known to have a potential antibacterialeffect14�26�27�38–40 and are suggested for therapy like wounddressing, burn injury8 etc. The degree of antibacterialactivity of AgNPs depends on different parameters likeshape, size and stability.22 There are several agents, whichhave been successfully employed to stabilize AgNPs andthe resulting AgNPs show different degree of activities.



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In present study we have used simple and cost effectivemethod to synthesize AgNPs and coating with some lessexpensive agents to prevent the agglomeration of AgNPs.It has been reported that PEG coated AgNPs are most sta-ble and effective.41�42 So we have included PEG in ourexperiments. The other two have also been reported asbetter functionalizing agents and also available easily.41

The successful coating was confirmed by various physicalmethods. Moreover antibacterial activities of each set offunctionalized nanoparticles were tested. A significant dif-ference in antibacterial activity was obtained when AgNPscoated with PEG was compared with AgNPs coated withT80 and SDS. There may be several reasons for increasedactivity of PEG coated AgNPs compared to the other two.The size of PEG coated AgNPs are smallest and thus theycan have relatively more chance to interact with bacteriaor they may have better access to internal bacterial pro-teins. To address how the size of AgNPs is related withantibacterial activity, we have synthesized different sizes ofcoated AgNPs. When the diameter of all the coated AgNPsare same, antibacterial activity of PEG coated AgNPs arehighest compared to the other two. The antibacterial activ-ity of SDS coated AgNPs are confined to the diameterrange 20 nm to 90 nm. Also SDS coated AgNPs haveleast antibacterial activity than the other two (Fig. 3(b)).Thus, despite having smallest size of PEG coated AgNPsin our study, additional features of PEG coated AgNPsmay be responsible for their antibacterial activity. Duringthe reduction process of silver salt, perhaps PEG is coatedwith AgNP in a better way compared to the other twofunctionalizing agents we have used. Moreover, release ofAg+ ions by AgNPs, as have been suggested,43 may alsocontribute significantly for antibacterial effect of AgNPs.So, it is likely that PEG coated AgNPs inside the bac-terial cellular environment release more Ag+ ions com-pared to the other two. We have also observed that PEGcoated AgNPs produce more ROS which may also beresponsible for antibacterial activity. These results are inwell agreement with previous report where it has beensuggested that Ag+ ions produces ROS inside the bac-terial cells.44�45 However, the exact mechanism, which isresponsible for killing wide range of bacteria, is not knowncurrently. The rapid breakdown of AgNPs release ionicsilver may inactivates vital bacterial enzymes by interact-ing with essential thiol groups. Ag+ ions can also inhibitbacterial DNA replication,46 damaging bacterial cytoplas-mic membranes, depleting levels of intracellular adenosinetriphosphate (ATP) and causing cell death. In our case, wehave repeatedly washed AgNPs to minimize the presenceof any free ions, specifically Ag+ ions. Further additionof N-acetyl cysteine (NAC) to AgNPs does not changethe antibacterial activity (data not shown), indicating theabsence of any free ions. We have additionally providedevidences that production of intracellular ROS may be oneof the possible mechanisms of cell killing. Due to large

surface to volume ratio, AgNPs particularly PEG coatedone, might have greater chances to enter into the cellswhere they can interact with cellular DNA/enzymes whichare essential for bacterial metabolisms or survival.45 In thatprocess AgNPs induced ROS ultimately block all the cel-lular function essential for survival or growth of bacteria.In some literature, AgNPs were reported to be cytotoxicto human cells. This may either due to the higher concen-trations of AgNPs used in those experiments or cells weretreated with AgNPs for longer period of time renderingmore Ag+ released inside the cells.47 This may depend onthe nature of functionalizing agents as well as the type ofcells used. Here also when we treated human cell lineswith coated AgNPs for longer period of time (48 h and72 h), significant cytotoxicity was observed (except SDScoated AgNPs) particularly for the PEG coated one. Thisis possible only when AgNPs can damage the cell mem-brane or they may enter into the cells to interact with spe-cific targets. But surprisingly when they were incubatedfor shorter time (18 h), they were non toxic or very lesstoxic to human cell lines.Though the mechanisms of action of AgNPs on bac-

terial cells remain elusive, it is obvious that they do notdepend on the resistance character of bacteria. The antibi-otic resistance character of bacteria occurs when the spe-cific biochemical pathway is blocked by antibiotics andbacteria generate enzymes to inactivate the antibiotics.Usually these enzymes are not synthesized in bacteria butexcessive amount of chronic exposure of antibiotics orantibiotic induced stressed condition may enforce the bac-teria to produce such antibiotic inactivating enzymes. Fromour study it seems that functionalized AgNP induced cell-killing mechanism is different from that of antibiotics,indicating AgNPs as a good candidate to kill antibioticresistant bacteria. Thus use of AgNP as an antibacte-rial therapeutic agent, particularly the PEG coated one ispromising and cost-effective.

LIST OF ABBREVIATIONS

PEG Polyethylene glycolT80 Tween 80SDS Sodium docyl sulfateAgN03 Silver nitrateNaBH4 Sodium borohydrideAgNPs Silver nanoparticlesSPR Surface plasmon resonanceTEM Transmission electron micrographDLS Dynamic light scatteringFTIR Fourier transform infrared spectroscopyROS Reactive oxygen speciesNBT Nitro blue tetrazoliumDMSO Di methyl sulphoxideMDR Multi-drug resistancePBS Phosphate buffer saline



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MIC Minimum inhibitory concentrationMBC Minimum bacteriocidal concentrationNAC N-acetyl cysteineHBBS Hank’s balanced salt solution

Acknowledgments: This work was supported by DST-PURSE programme of Jadavpur University and StateGovt, Fellowship Scheme of Jadavpur University, WestBengal, India. Assistance from the Central instrumentsfacility from IACS and NICED (West Bengal India) forFTIR, TEM analysis is gratefully acknowledged.

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Received: 1 September 2011. Accepted: 16 January 2010.


Antibacterial Activities of Polyethylene Glycol, Tween 80 and Sodium Dodecyl Sulphate Coated Silver...

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